The longer term goal of the research is aimed at finding new ways to prevent or to treat the infection caused by respiratory syncytial virus (RSV). In order to reach such goal, it is required to study the molecular aspects by which the virus enter to the cell, and how the RSV could take advantage of this to modulate the immune response. RSV has been proposed to fuse its envelope directly with the cell membrane;however, cell membrane is not a homogeneous place and that it is constituted by different microdomains such as lipid rafts. The current proposal would help clarify the role that lipid rafts are playing during the viral penetration and the mechanistic aspects involved for allowing the lipid raft to participate. Since lipid rafts cluster very important signaling molecules involved in both innate and adaptive immunity, this proposal would also test the hypothesis that RSV proteins target lipid rafts to modulate the immune response. The specific aims of this project are: 1) To determine whether lipid raft trafficking constitutes a very important pathway for RSV to infect normal human bronchial epithelial (NHBE) cells. To address this aim, lipid rafts will be disrupted by taking away the free membrane cholesterol and RSV infection will be examined in such context. The RSV envelope protein targeting the lipid raft will be examined by evaluating whether RSV-deficient/delta for G and/or SH use lipid rafts to infect NHBE cells. 2) Determine which signaling pathway is involved in assembly of lipid rafts triggered by RSV infection. The role of PKC-alpha, calcium, PI-PLC, and dynamin-2 in the lipid raft clustering triggered by RSV will be examined using pharmacological inhibitors, and dominant negative constructs. 3) Determine whether RSV targets lipid rafts in order to modulate the immune response. NHBE cells transfected with plasmids encoding both EGFP and secreted G (sG) protein will be co-cultured with non-transfected NHBE cells. ERK-1/2 and STAT-1 signaling will be examined using EMSA and anti-phospho-specific antibodies for western-blots and immunocytofluorescence. The effect of the potential presence of RSV proteins in the immunological synapse on the dendritic cell side will be examined by immunofluorescence and confocal microscopy. In Colombia, RSV is the most important respiratory pathogen agent causing 41.8% of the new cases of lower tract respiratory illness in infants. Understanding the mechanistic process of the RSV infection will help in the development of the vaccines and therapies.